Abstract: Objective To determine the three-dimensional(3D) conformation of staphylococcal protein A-gold(SPA-G) single molecule using atomic force microscope(AFM) so as to evaluate the feasibility of using this molecule for in situ labeling of the neuronal membrane protein. Methods AFM was used to acquire the images of SPA-G binding to the surface of mica under physiological condition for determining the 3D conformation of the molecule. Results SPA-G single molecule was shown to contain a characteristic structure with a chain in the shape of mirror image of the letter C, which had the dimension of 48.80 nm ×42.13 nm×20.53 nm. Conclusion AFM provide a new means for morphological investigation of the biomacromolecule at the nanometer scale in physiological conditions, and SPA-G can be utilized for in situ labeling of the neuronal membrane receptors.